Abstract
Tuberculosis (TB) is an ancient infectious disease of man and animals caused by Mycobacterium spp and responsible for considerable morbidity and mortality each year. TB in man and animals is mostly caused by Mycobacterium bovis (M. bovis, bovine TB), Mycobacterium tuberculosis (M. tuberculosis, human TB) and Mycobacterium avium sub. sp var paratuberculosis (M. aviun, para TB). This work was aimed at the rapid and specific identification of the etiology of TB in cattle by using intradermal tuberculin test, gross and microscopic examination, Ziehl-Neelson staining of smears and tissue sections and culture of Mycobacterium on Lowenstein-Jensen (L-J) media. The suspected cases of human TB were investigated by using Ziehl-Neelson staining of smears and culture of bacteria on L-J media. Attempt was taken for the adoption/designing polymerase chain reaction (PCR) for the rapid and accurate detection of the fragments of 16srRNA, MPB83, H37Rv, H37Rv Rv3479HP and AN5 genes for specific identification of the etiology of TB. Out of 696 dairy cattle tested using intradermal tuberculin test in the selected Government and non-government dairy cattle farm of Bangladesh, 23 yield hypersensitivity reactions to tuberculin. Holstein Friesian cross breed cattle (n=21) appeared more susceptible to clinical forms of TB than other breed of cattle. A total of 11 tuberculin test positive cattle were investigated for further analysis. Aspiration biopsies from the superficial lymphnodes of suspected cattle were smeared on to clean slides stained with Zeihl-Nelson staining and showed acid fast bacteria in 05 cases. Results of gross and microscopic analyses showed that 10 cattle had systemic TB and one had TB in myeloid form. The bacteria from 11 cattle samples were, therefore, grown on L-J media for a period of 06 weeks and observed bacterial growth in 07 cases. Results of uniplex and multiplex PCR with the extracted DNA from 11 cases showed that 09 cattle were infected with bovine TB and two were with human TB. Two cattle were co-infected with bovine TB and para TB. A total of 18 suspected cases of human TB were examined (sputum, pleural fluid and peritoneal fluid). Smears from these samples on to clean slides were stained with Zeihl-Nelson staining and showed Mycobacterium in six cases. Genomic DNA were extracted from the sputum, pleural and peritoneal fluid of 18 cases and uniplex and multiplex PCR (mPCR) was carried. Out of 18 suspected cases 11 showed specific amplicons in PCR settings. Results of PCR showed that the suspected human were infected with bovine TB (N=02) and human TB (N=09). Para TB was detected with two human cases. Sequencing and phylogenetic analysis of the selected genes of bovine and human TB showed that the organisms investigated were belonging to Lineage 1, corresponds to the East African-Indian (EAI) family. Analyses of partial sequence of genes also demonstrate that the Mycobacterium of bovine and human TB in Bangladesh is genetically similar with the TB strain currently circulating in neighboring country. In conclusion the organized dairy cattle of Bangladesh and human being of Mymensingh district were infected with M. bovis, M. tuberculosis and Mycobacterium avium sub. sp var paratuberculosis. The PCR test protocol I have adopted and designed showed higher sensitivity and specificity than acid fast staining and cultural identification and can be used to detect TB in man and animals. Bovine TB, human TB and para TB are extremely zoonotic pathogens, endemic in cattle and human in Bangladesh. It needs to perform routine test on to the cattle of old dairy farms using tuberculin test and PCR and dispose the infected or carrier at early onset to prevent further zoonosis.